The excitatory amino acid transporters (EAATs) clear glutamate from the synaptic cleft following rounds of neurotransmission and are responsible for the uptake of acidic and neutral amino acids by all cell types. Driven by pre-existing electrochemical gradients, EAATs couple substrate uptake to the co-transport of sodium ions and protons and to the counter-transport of potassium. Previous crystallographic studies on a bacterial homologue from Pyrococcus horikoshii, GltPh have revealed the transporter in the outward facing states with the substrate-binding site accessible to the extracellular solution and either occluded when bound to a substrate or exposed when bound to a blocker. These structures have explained how the substrate and sodium ions reach their binding sites from the extracellular solution but left unanswered how these solutes are translocated across the membrane and released into the cytoplasm. It is believed that a conformational transition of the transporter into an inward facing state, in which the substrate-binding site is accessible to the intracellular solution, is a prerequisite for the substrate dissociation into the cytoplasm. In the current application, we propose to use double cysteine mutagenesis and cross-linking to delineate the structural re- arrangements that occur upon transition of the transporter into the inward facing state and to probe the dynamics of the process. We further propose to determine the crystal structures of the transporter molecules covalently constrained in the inward facing state and possibly in other intermediate transport states by cysteine cross-linking. Finally, we will employ the site directed mutagenesis in conjuncture with biochemical and structural analyses to probe the location of the ion binding and permeation sites in GltPh and, by analogy, in EAATs PUBLIC HEALTH RELEVANCE: Glutamate transporters are membrane proteins responsible for the clearance of the neurotransmitter glutamate from the synapses following rounds of neurotransmission. Their malfunction is associated with numerous diseases and pathological states including neurodegeneration, epilepsy, schizophrenia, traumatic brain injury and stroke. We focus our study on the mechanism and atomic structure of these molecular pumps essential for the proper development and function of the central nervous system.